Screening of the Proteins Interacting with the HDAC1in Yeast Two-hybird System
|School||Dalian University of Technology|
|Keywords||HDAC1 Yeast Two-Hybird co-immunoprecipition|
The innormal acetylation of histones mediated by histoneacetylase (HATs) andhistonedeacetylase (HDACs) is the most typical way of the activation of tumors. It isnecessary to screen the protein in cDNA library of HeLa genomic that can interacted withHDAC1in order to looking for a novel target of anticancer drug preferably. In this thesis, theplasmid pGBKT7-HDACl was constructed and used for the seclection of target prptein inyeast two-hyberid system.The total RNA was extracting from HeLa cancer cell by trizol method and cDNA wasreverse-transcribe using Invitrogen Reverse Transcription Kit according to the protocol of themanufacture. The complete coding region array of HDACl gene was amplifyed by PCR. Thefragment of HDACl was constructed into the eukaryotic expression vector pGBKT7-HDAClby recombining DNA technology, the expression of which in AH109cells was confirmed bydetection of nontoxic and nonself-activated on yeast AH109. The yeast AH109was mixed andfused with the yeast Y187containing Human HeLa cDNA library for screening interactionalproteins. The cotransfection of the recombinant HDAC1plamsid and recombinant FHL2plamsid into293FT cells was confirmed by the method of immuno-coprecipitation, whichsuggested their effects betwen HDAC1and FHL2.Successful construction of pGBKT7-HDACl recombinant plasmid and expression ofpGBKT7-HDACl recombinant plasmid in prokaryotic cell that was conformed by westernblot. the results of immuno-coprecipitation test indicated that the protein called FHL2caninteract with HDAC1, but the negative control can not bind with it, which suggested thatHDAC1can be linked to FHL2specifically.Constructed prokaryotic expression vector pGBKT7-HDAC containing fragmentHDAC1can express HDAC1protein in AH109cell, which means at the protein level ofFHL2regulated histone deacetylase. It is concluded that HDACl maybe another ways tomodulate FHL2and play a cirtical role in the occurrence, development and prognosis ofcancer.